The long-term objective for this continued application is to identify the role of beta cell autoantigens in insulin-dependent diabetes mellitus (IDDM). Our previous research resulted in the discovery of the 64K antigen in IDDM and the cloning of this antigen to demonstrate that human islet beta cells express a novel glutamic acid decarboxylase (GAD) isoform, GAD65. Using recombinant human GAD65 we show that the GAD65 autoantibodies (GAD65Ab) in IDDM are isoform-specific and that GAD65 is primarily expressed in the beta cells. The GAD65 cDNA was used to develop a novel radiobinding assay using 35S- or 3H-GAD65 prepared by in vitro coupled transcription translation (IVTT). In an international standardization, this GAD65 assay has the highest diagnostic sensitivity (82%) and specificity (98.8%) for IDDM. IDDM and Stiff Man Syndrome patients share the GAD65 autoantigen but the antibody epitopes are shown to differ. The aims are to determine the GAD65Ab epitopes in IDDM by analysis of IVTT GAD65/GAD67 chimeric molecules and by using site-directed mutagenesis to generate GAD65 mutants at the putative epitope 440-570 amino acid region of GAD65. The role of the epitope-specific GAD65 autoreactivity in IDDM will be tested in the following specific aims: 1) To determine, a) IDDM antibody epitopes in radiobinding assays with GAD65/67 chimeras and GAD65 mutants, b) IgM, IgA and IgG epitope- specific antibodies in IDDM and controls, and c) the sensitivity and specificity of epitope-specific GAD65Ab for IDDM in a large population- based study of IDDM patients and controls as well as in the Seattle prospective family study. 2) To test the hypothesis that IDDM epitope- specific GAD65Ab have heavy and light chain sequences which are unique to IDDM. We will, a) construct combinatorial antibody libraries from new onset IDDM patients to isolate Fab reactive GAD65, b) determine cDNA sequences of heavy and light chain anti-GAD65 Fab selected by GAD65 epitope-specific panning; and c) transfect anti-GAD65 Fab to human B cells to explore the role of antibody fine-specificity in influencing epitope presentation. Finally: 3) to test the hypothesis that epitope- specific GAD65Ab mark the specific loss of beta cells in IDDM, we will determine, a) the full length GAD3 sequence of the 2.5-kb transcript present in human and monkey islets and compare it with GAD1 (3.7-kb transcript- not in islets) and human islet GAD2 (5.6-kb transcript), b) IDDM antibody reactivity of the putative GAD3 protein tested in our radiobinding IVTT assay, and c) GAD1, GAD2 and GAD3 gene expression during development as well as during insulitis. This research will continue to dissect IDDM autoreactivity to islet cell autoantigens and define the pathogenetic mechanisms which may provide insights into the possible prevention and cure of this disease.